Lin M, Hultquist K L, Oh D H, Bauer E A, Hoeffler W K
Department of Dermatology, Stanford University School of Medicine, California 94305, USA.
FASEB J. 1995 Oct;9(13):1371-7. doi: 10.1096/fasebj.9.13.7557028.
Type I collagenase plays an important role in both tumor metastasis and the remodeling of connective tissue in normal human skin, during wound healing, for example, and may participate in the pathophysiology of some dermatological diseases such as skin cancer and a chronic blistering disease, recessive dystrophic epidermolysis bullosa. In an effort specifically to inhibit collagenase expression, we have designed phosphorothioate antisense oligonucleotides, linked at the 5' ends with photoreactive 4'-(hydroxyethoxymethyl)-4,5',8-trimethyl-psoralen (HMT), and directed them against the 5' end of the collagenase mRNA. Two antisense-HMT molecules targeting a region overlapping the initiation codon were compared. Only one contained the HMT moiety targeting a 5'TpA on its complementary sense strand, and we observed greater than 50-fold improvement on the cross-linking of this antisense oligonucleotide to its target sequence after ultraviolet A (UVA) irradiation. Likewise, sequence complementary to the 5'TpA target was also required to demonstrate specific inhibition of in vitro translation of collagenase mRNA. Tissue culture experiments, conducted by incubation of collagenase-specific antisense-HMT oligonucleotides with fibroblasts in monolayer or in 3-dimensional dermal equivalents, showed lowered collagenase levels 24 h after UVA irradiation as compared to controls. Initial screening of antisense oligomers for specific hybridization and photo-cross-linking is a useful step in the design of antisense oligonucleotides, and allowed us to design an HMT-linked antisense phosphorothioate oligonucleotide that specifically inhibits the expression of fibroblastic collagenase.
I型胶原酶在肿瘤转移以及人体正常皮肤结缔组织重塑过程中发挥着重要作用，例如在伤口愈合期间，并且可能参与某些皮肤病的病理生理过程，如皮肤癌和一种慢性水疱病——隐性营养不良性大疱性表皮松解症。为了特异性抑制胶原酶的表达，我们设计了硫代磷酸酯反义寡核苷酸，其5'端与光反应性4'-(羟乙氧基甲基)-4,5',8-三甲基补骨脂素（HMT）相连，并将其靶向胶原酶mRNA的5'端。比较了两种靶向与起始密码子重叠区域的反义-HMT分子。只有一种在其互补有义链上含有靶向5'TpA的HMT部分，并且我们观察到在紫外线A（UVA）照射后，该反义寡核苷酸与靶序列的交联有超过50倍的改善。同样，与5'TpA靶标的序列互补也被要求用于证明对胶原酶mRNA体外翻译的特异性抑制。通过将胶原酶特异性反义-HMT寡核苷酸与单层成纤维细胞或三维真皮替代物一起孵育进行的组织培养实验表明，与对照相比，UVA照射24小时后胶原酶水平降低。对反义寡聚物进行特异性杂交和光交联的初步筛选是反义寡核苷酸设计中的一个有用步骤，并且使我们能够设计出一种特异性抑制成纤维细胞胶原酶表达的HMT连接的硫代磷酸酯反义寡核苷酸。
